Methods for treating valve-associated regions of vascular vessels

ABSTRACT

This invention relates in one aspect to the treatment of a valve-associated region of a vascular vessel with a biomaterial. The biomaterial can be a remodelable material that strengthens and/or supports the vessel walls. Additionally the biomaterial can include a variety of naturally occurring or added bioactive agents and/or viable cellular populations.

REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/624,775 filed Nov. 3, 2004, pending, which ishereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

In general, the present invention relates to methods of treatingpatients suffering from defects or diseases of the vascular system. Morespecifically, the present invention is directed to methods for treating,strengthening, and/or repairing vascular vessels in regions in andaround internal valve structures.

A variety of vascular disorders arise in and around the site of vascularvalves. One common disorder is venous valve insufficiency resulting fromvalvular dysfunction, most often occurring in the legs. The valves inthe veins do not adequately inhibit reflux or retrograde blood flow.Varicose veins resulting from venous valve insufficiency can be quitepainful for the patients who suffer from this condition and can alsocause embarrassment over the appearance of the varicose veins. In moresevere cases, venous valve insufficiency can lead to general swelling inthe patient's lower extremities, and can ultimately result in venousstasis ulcers of the skin and subcutaneous tissue. While there is nocure for these conditions, a variety of treatment methods have been usedor suggested. One treatment method includes resecting the affected veinsegment and replacing the resected vein with an autologous vein removedfrom the patient or by reconstructing venous valves. Other treatmentmethods include placing constricting sutures around the veins; veinablation, where whole sections of the veins are closed off usually viaheat or chemical treatment; and ambulatory phlebectomy, where theabnormal vein is removed. Less invasive treatments include wearingcompression stockings about the patients legs to physically compress theveins and tissue surrounding the veins.

There is a continuing need for advancements in the relevant field,including improved and/or alternative treatment methods and devices. Thepresent invention is addressed to these needs.

SUMMARY

In various aspects, the present invention relates to the treatment ofpatients with vascular diseases and the use of implantable devicestherefor. While the actual nature of the invention covered herein canonly be determined with reference to the claims appended hereto, certainforms and features, which are characteristic of the preferredembodiments disclosed herein, are described briefly as follows.

In one form, the present invention provides a method for treating a veinin a patient. The method comprises accessing a treatment site proximateto a valve in the vein, and introducing a remodelable biomaterial intoor externally of a wall of the vein at the treatment site. For example,such methods may involve treating a venous defect associated with avenous valve in a patient, e.g. by reinforcing and/or re-shaping aweakened or distended vein wall. The remodelable biomaterial cancomprise a collagen-based material such as an extracellular matrixmaterial. Submucosa tissue or another similar collagenous layers can beused. In selected embodiments, the remodelable material can alsofunction as a matrix that includes added or naturally occurringbioactive agents and/or viable cells. The remodelable biomaterial can beconfigured into a three dimensional construct for a prosthesis to beimplanted into a patient. When the prosthesis is implanted into atreatment site the biomaterial can shrink. This propensity to shrink canbe used to support and/or modify (e.g. constrict) the vessel walls.

In another form, the present invention provides a method for treating avein of a patient. The method comprises accessing a treatment site alongthe vein, said treatment site proximate to a valve within the vein, andintroducing a flowable mass of biocompatible material in contact with anexterior surface of a wall of the vein at the treatment site. In certainembodiments, the biocompatible material is remodelable biomaterial. Thetreatment site can be selected to be adjacent to the base of one or moreleaflets of the valve, or upstream or downstream of the base of thevalve leaflet(s). In certain embodiments of the invention, a remodelablebiomaterial can be provided in injectable form, and can be injected to alocation exterior of the vein wall at or adjacent to its intersectionwith the leaflet base(s). Such treatments can, for example, be used tomodify the lumen of the vein, e.g. to bulk at the exterior of adistended vein segment and drive the vein walls inwardly so as to driveleaflets toward one another to improve valve function. In otherinventive modes, such treatments can, for example, be used to providereinforcement to the vein wall to inhibit the onset or continuation ofwall distention. Such reinforcement can be provided at any suitableregion exterior of the vein wall proximate to a venous valve, includinglocations upstream of the base of the leaflets, at the base of theleaflets, or downstream of the leaflets, for example within andpotentially beyond the region of the valve sinus.

In another form, the present invention provides a method of treating avascular defect in a patient associated with a vascular valve. Themethod comprises locating such a vascular defect in the patient; andinjecting an injectable submucosa-containing composition into atreatment site proximate to the vascular defect. The submucosacomposition can be in fluid form when injected and induced or allowed togel or solidify in vivo. The submucosa-containing composition caninclude a number of different added or naturally occurring bioactiveagents and viable cells.

In yet another embodiment, the present invention provides a method fortreating a patient, wherein the method comprises treating a nativevascular valve and/or introducing an artificial vascular valve in avascular vessel of the patient. The method further includes reinforcingthe vascular vessel in a region proximate to said native vascular valveor artificial vascular valve. Reinforcement may be provided byintroducing a biocompatible material external of the vein or othervessel. The biocompatible material can be a remodelablecollagen-containing material such as an extracellular matrix material.The remodelable material can be introduced in a solid form, e.g.configured into a three dimensional construct for a prosthesis to beimplanted into the patient around the vessel, or in a flowable form,e.g. as in a flowable ECM composition that gels after introduction.

Further objects, features, aspects, forms, advantages and benefits shallbecome apparent from the description and drawings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a cross-section of a vein and an artery.

FIG. 2 is a cross-sectional view of a healthy vein showing a pair ofvalve leaflets.

FIG. 3 is a cross-sectional view of a vein exhibiting one manifestationof valvular dysfunction.

FIG. 4 illustrates one embodiment of a construct formed of a sheet of abiomaterial in accordance with the present invention.

FIG. 5 is an illustration of an alternative embodiment of a constructformed of a sheet of a biomaterial in accordance with the presentinvention.

FIG. 6 illustrates the positioning of the prosthesis of FIG. 4 about avein in accordance with the present invention.

FIG. 7 illustrates the positioning of the prosthesis of FIG. 5 about avein in accordance with the present invention.

FIG. 8 provides an illustration of a tissue volume containing a vein andin which an injected mass of biomaterial forms a cuff around the vein.

FIG. 9 provides a cross-sectional view illustrating the injection of amass of material to surround a vein in the region of a valve.

FIG. 10 provides a cross-sectional view illustrating the injection of amaterial into the vein wall in the region of a valve.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the invention, certaintreatment methods, prosthesis devices, and materials will be discussed.It will nevertheless be understood that no limitation of the scope ofthe invention is thereby intended. Any alterations and furthermodifications in the described materials, prostheses, and treatmentmethods, and any further applications of the principles of the inventionas described herein, are contemplated as would normally occur to oneskilled in the art to which the invent relates.

The present invention provides methods, devices and materials fortreating vascular vessels at sites associated with vascular valves.Vascular vessels, including veins and arteries, are prone to a varietyof diseases and defects that affect the function, strength, andintegrity of the vessels in regions associated with valves within thevessels. The treatment according to the present invention can includeintroducing a biomaterial into a treatment site located in tissueadjacent the vessel and/or in the vessel's walls. The biomaterialprovides a benefit to the vascular vessels, for example to treat defectsand diseases as described more fully herein. The materials and methodsdescribed herein are advantageous for treatment of disorders associatedwith venous valves, particularly those occurring within limbs such aslegs. Further, the biomaterial can be a remodelable material and mayinclude one or more bioactive substances derived from the source of thebiomaterial and/or added to the biomaterial. The biomaterial can also beseeded with or contain viable cells.

For the purposes of certain aspects of the descriptions herein, FIG. 1illustrates a cross-sectional view of a normal vein and an artery.Artery 10 is composed of three coats: an internal or endothelial coat(tunica intima) 12; a middle muscular coat (tunica media) 14, and anexternal connective tissue coating (tunica adventitia) 16. Typically,the two inner coats 12 and 14 together are very easily separated fromthe external coating. The vein 18 is also composed of threecoats—internal, middle, and external coats 20, 22, and 24, respectively.A significant structural difference between veins and arteries is thecomparative weakness of the middle coat of the vein. Because of this,the veins do not stand open when divided as the arteries do.Consequently, a vein tends to collapse without an internal fluid orfluid pressure, while a healthy artery substantially retains its naturalshape and relative dimension regardless of whether or not it isconducting blood (or other fluid). Additionally, many veins, includingthe larger veins in the lower extremities, contain multiple valves toprevent the reflux or retrograde flow of blood away from the heart andthereby facilitate the return of blood to the heart.

“Remodeling” as used herein generally refers to the production of hosttissue that replaces an implanted material. The development of hosttissue can occur at a functional rate about equal to the rate ofbiodegradation of the implanted material, resulting in a replacement ofthe implanted material by host cells.

In certain embodiments, the present invention provides a treatment forvascular vessels using a prostheses formed of a biomaterial containingconstruct. It is preferred to use a remodelable biomaterial that canalso serve as a biocompatible scaffold with the ability to remodel hosttissue. Accordingly, a naturally occurring biomaterial is highlydesirable. A biomaterial for use in the present invention can beobtained as a purified collagen-based matrix structure.

One such collagen-based biomaterial is extracellular matrix (ECM).Preferred are naturally-derived collagenous ECMs isolated from suitableanimal or human tissue sources. Suitable extracellular matrix materialsinclude, for instance, submucosa (including for example small intestinalsubmucosa, stomach submucosa, urinary bladder submucosa, or uterinesubmucosa, each of these isolated from juvenile or adult animals), renalcapsule membrane, amnion, dura mater, pericardium, serosa, peritoneum orbasement membrane materials, including liver basement membrane orepithelial basement membrane materials. These materials may be isolatedand used as intact natural sheet forms, or reconstituted collagen layersincluding collagen derived from these materials and/or other collagenousmaterials may be used. For additional information as to submucosamaterials useful in the present invention, and their isolation andtreatment, reference can be made to U.S. Pat. Nos. 4,902,508, 5,554,389,5,993,844, 6,206,931, and 6,099,567. Renal capsule membrane can also beobtained from warm-blooded vertebrates, as described more particularlyin International Patent Application serial No. PCT/US02/20499 filed Jun.28, 2002, published Jan. 9, 2003 as WO03002165.

The biomaterial can retain growth factors or other bioactive componentsnative to a source tissue. For example, submucosa or other ECM materialsmay include one or more growth factors such as basic fibroblast growthfactor (FGF-2), transforming growth factor beta (TGF-beta), epidermalgrowth factor (EGF), and/or platelet derived growth factor (PDGF). Aswell, submucosa tissue used in the invention may include otherbiological materials such as heparin, heparin sulfate, hyaluronic acid,fibronectin and the like. Thus, generally speaking, the submucosa orother ECM material may include a bioactive component that induces,directly or indirectly, a cellular response such as a change in cellmorphology, proliferation, growth, protein or gene expression.

The biomaterial can be used alone, or in combination with one or moreadded pharmacologic agents, such as physiologically compatible minerals,growth factors (including vascular endothelial growth factors),antibiotics, chemotherapeutic agents, antigens, antibodies, geneticmaterial, enzymes and hormones and the like.

In certain forms, the biomaterial of the invention can also be used incombination with other nutrients which support the growth of cells, e.g.eukaryotic cells such as endothelial (including non-keratinized orkeratinized epithelial cells), fibroblastic cells, smooth muscle cells,cardiac muscle cells, multipotent progenitor or stem cells, pericytes,or any other suitable cell type (see, e.g. International Publication No.WO 96/24661 dated 15 Aug. 1996, publishing International Application No.PCT/US96/01842 filed 9 Feb. 1996). Such cells may optionally be includedin the biomaterial construct or composition, for example being seededupon or incorporated within the biomaterial construct or composition.The submucosa or other biomaterial substrate composition can beeffective to support the proliferation and/or differentiation ofmammalian cells, including human cells. Still further, the inventivemethods herein may use a biomaterial that serves as a matrix that cansupport and produce genetically modified cells, (see, e.g.,International Publication No. WO 96/25179 dated 22 Aug. 1996, publishingInternational Application No. PCT/US96/02136 filed 16 Feb. 1996; andInternational Publication No. WO 95/22611 dated 24 Aug. 1995, publishingInternational Application No. PCT/US95/02251 filed 21 Feb. 1995). Suchcompositions for genetically modifying cells can include an ECM such assubmucosa or another collagenous biomaterial as a three dimensionalconstruct or a fluidized or flowable material in combination with anucleic acid molecule containing a sequence to be expressed in cells,e.g. a recombinant vector such as a plasmid containing a nucleic acidsequence with which in vitro or in vivo target cells are to begenetically modified.

It some forms of practicing the invention, the biomaterial according tothe present invention is substantially free of any antiviral agents orany antimicrobial-type agents, which can affect the biochemistry of thebiomaterial and its efficacy upon implantation. One method of treatingtissue material is to rinse the delaminated tissue in saline and soak itin an antimicrobial agent, for example, as disclosed in U.S. Pat. Nos.4,956,178 and 6,666,892. While such techniques can optionally bepracticed with isolated collagenous ECMs such as submucosa in thepresent invention, preferred embodiments avoid the use of antimicrobialagents and the like, which not only can affect the biochemistry of thecollagenous biomaterial but also can be unnecessarily introduced intothe tissues of the patient.

Irrespective of the origin of the biomaterial, a prosthesis for use inthe present invention can be made thicker by making multilaminateconstructs, for example SIS constructs or materials as described in U.S.Pat. Nos. 5,968,096; 5,955,110; 5,885,619; and 5,711,969; thedisclosures of which are entirely and expressly incorporated byreference. The layers of the laminated construct can be bonded togethervia a crosslinking agent, a bonding agent, dehydrothermal bonding and/orby use of biotissue welding technique(s), or other suitable means.

In certain embodiments, the ECM or other biomaterial can be initiallyprocessed to provide a predetermined, three-dimensional shape orconstruct, which will be implanted into the patient as a prosthesis andwill substantially retain its shape during remodeling or replacement ofthe graft with endogenous tissues. Illustratively, in embodimentswherein a sheet-form collagenous biomaterial is provided partially orcompletely encircling a vein or artery, the biomaterial can be processedto have the shape of a complete or partial cylinder configured to fitaround and contact the external walls of the vessel about which theconstruct is to be received. Processing to provide such shapes caninclude configuring the ECM or other biomaterial to a desired shapewhile fully or partially hydrated, and dehydrating the material while insuch shape. Such dehydrating may for example be conducted by air drying,lyophilization, vacuum pressing, or other suitable techniques.

For treatment of blood containing vessels in accordance with the presentinvention, and particularly for situations in which exposure of anamount of the biomaterial to the interior of the vessel is needed or arisk, the biomaterial can be treated to reduce any thrombogeniccharacter that it may have. In this regard, an antithrombotic agent,such as, heparin or a heparin derivative may be bound to thebiomaterial, construct or a prosthesis formed from the construct by anysuitable method including physical, ionic, or covalent bonding. Forexample, this may be accomplished for example by applying solution ofheparin or a heparin derivative to the biomaterial's surface or bydipping the biomaterial in the solution. In one embodiment, heparin isbound to the biomaterial using a suitable crosslinking agent such as apolyepoxide or carbodiimide cross linking agent such as1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). Inmulti-layer constructs, heparin or other agents can be applied to thelayers individually before incorporation of the layer into theconstruct, after the layers are incorporated into the construct (e.g.coating a luminal surface of an inner tubular layer), or both. Heparincan also be applied using a benzalkonium heparin (BA-Hep) isopropylalcohol solution. This procedure treats the collagen with an ionicallybound BA-Hep complex. Other coating, bonding, and attachment procedures,which are known in the art can also be used. As well, in the case offlowable (e.g. injectable) biomaterials, heparin or one or more otheranti-thrombogenic agents may be incorporated into the formulationutilized in soluble form and/or bound to any suspended particulatebiomaterial within the formulation.

The present invention provides for the treatment of vascular defectsassociated with vascular valves. In preferred embodiments, the presentinvention provides for the treatment of vascular defects associated withvenous valves. Illustratively, FIG. 2 shows a cross-sectional view of aportion of normal vein 30 with the bicuspid venous valve illustratedgenerally as valve 32. Valve 32 includes a pair of opposing leaflets 34and 36. Each leaflet 34 and 36 extends from a base 38 on the internal orendothelial coating 40 toward the center of vein 30 terminating in agenerally convex cusp. Valve 32 generally allows blood to flow in onlyone direction toward the heart i.e., antegrade blood flow (see arrow).In normally functioning veins, during antegrade blood flow, the leafletslie close to or against the internal wall of the vein typically recessedwithin a sinus cavity 42 formed in the vein 30 such that the overallinternal diameter of the vein is not significantly diminished by thepresence of the leaflets. However, during retrograde blood flow, theleaflets 34 and 36 are forced away from the sinus cavity walls such thatthe cusps of the leaflets extend into the central portion or channel ofthe vein 30. In healthy valves, which function to effectively inhibitretrograde blood flow, the leaflets co-apt or meet thus preventing theretrograde blood flow. However, in veins exhibiting valvulardysfunction, the opposing leaflets do not meet sufficiently to close offretrograde blood flow. This may be symptomatic of a variety of defectsor problems. Most commonly, the vein wall in the region of the leafletshas distended such that the internal diameter of the vein around theleaflets has increased, and therefore the leaflets cannot meet withsufficient efficacy to prevent substantial retrograde blood flow.

FIG. 3 is an illustration of a vein 50 exhibiting the manifestation ofvalvular dysfunction, which can be treated according to the presentinvention. Vein 50 includes the bicuspid valve 52. It can be seen fromthe illustration that the free edges of the individual leaflets 54 and56 do not meet together to inhibit retrograde blood flow. As notedabove, one manifestation of venous valve insufficiency is a condition inwhich the diameter of the vein has become enlarged, particularly aroundthe base of the leaflet. In this particular condition, since thecircumference of the vein at the base of the leaflet has expanded, theopposing cusps of the leaflets do not meet in the central channel of thevein. In certain embodiments of the invention, such a diseased ordefective region of the vascular vessel is identified in the patient,and is accessed for treatment. This access can be provided, for example,using surgical cut-down procedures, or using minimally invasiveprocedures such as percutaneous and/or endoscopic techniques. Fortreatments of the invention including the implantation of biomaterialsexternal of the vascular vessel, exposure of or other access to theentire external circumference of the vascular vessel, or only to one ormultiple points around the circumference of the vessel, may be utilized.

FIG. 4 is an illustration of one embodiment of a construct 70 to form aprostheses in accordance with the present invention. Construct 70 isformed of a remodelable biomaterial, and in one particular embodimentconstruct 70 is formed from submucosa. Construct 70 is illustrated as anelongated, planar construct, preferably in the form of a flexible sheet.Construct 70 can be composed of a single layer or of several layers as alaminate of the remodelable biomaterial. Construct 70 can be sized andshaped either prior to or during surgery to approximate at least aportion of the external circumference of a targeted vessel. Furtherconstruct 70 can be prepared to include one or more bioactive agentsand/or cellular populations depending upon the desired treatment regime.

In use, once the treatment site has been accessed sufficiently,construct 70 is wrapped about the external circumference of the targetvessel overlaying the treatment site. Construct 70 can partly orcompletely encircle the vessel. The first and second ends 72 and 74 ofthe construct 70 can be sutured or secured to the external circumferenceof the vessel wall in certain embodiments. In other embodiments,construct 70 is sized and formed such that the two opposite ends 72 and74 touch or overlap each other. The two edges can be secured togetherusing conventional means including suturing to each other (and/or to thevessel wall), tissue welding (see e.g. U.S. Pat. No. 5,156,613), and/orbonding agents such as fibrin glue, or other suitable techniques. Inthis regard, FIG. 6 provides an illustration of construct 70 receivedand secured with sutures around a vein 76 in the region of an interiorvalve 78 (partially shown in phantom). In particular, the illustratedconstruct 70 covers the region at which the leaflet base and vein wallintersect, and can be used to support the vein wall against initial orcontinued distention and/or to re-shape the vein so as to improve thefunction of the valve 78, for example by bringing the leaflet bases ofthe valve 78 closer together and improving the co-aptation of theleaflets.

FIG. 5 illustrates an alternative embodiment of a construct 80 for usein the present invention. Construct 80 includes a slot 82 formed at itsfirst end and a corresponding tab 84 extending from a second end. Inuse, tab 84 can be inserted into slot 82 to provide a generally circularcuff or band that can be placed around a vein or artery. Additionally,the ends, including tab 84, can optionally be secured together asdiscussed above for construct 70. FIG. 7 provides an illustration ofconstruct 80 received and secured around a vein 86 in the region of aninterior valve 88 (partially shown in phantom). In particular, as withthe construct 70 illustrated in FIG. 6, the illustrated construct 80covers the area of the vein wall corresponding to the base of theleaflets. This positioning can be used to support the vein wall againstinitial or continued distention and/or to re-shape the vein so as toimprove the function of the valve 78, for example by bringing theleaflet bases of the valve 78 closer together and improving theco-aptation of the leaflets. It will also be understood that construct80 can include plurality of tabs and corresponding number of slots tofacilitate securing the construct around a vein or other vascularvessel.

Although FIG. 6 and FIG. 7 show the constructs 70 and 80 positionedabout the exterior circumference of the vessel proximate to the base ofthe leaflets, it will be understood that other regions may also betreated in accordance with the invention. The prosthesis can bepositioned directly at or about the base of the leaflets or below thebase of the leaflets, i.e., further from the heart. Alternatively, theprosthesis can be positioned above the base of the leaflets, i.e.,closer to the heart. In other embodiments, two or more prostheses can beused, for example, one placed above and another placed below the base ofthe leaflets. In still other embodiments, the prosthesis can include areinforcing clamp or clip received around the construct 70 or 80. Thereinforcing clamp or clip can provide further support and compressagainst the external side walls of vessel.

The thickness of the construct(s) to form the prosthesis can be selecteddepending upon the tissue or biomaterial used, its intended use, and/ora prescribed treatment regime. In preferred embodiments for thetreatment of veins and arteries, the biomaterial can be provided in athickness of between about 50 and about 500 microns. It will beunderstood that the thickness of the construct/prosthesis can also bevaried or, in particular, increased by laminating two or more layers ofa biomaterial onto one another. It will also be understood that otherconfigurations of the prosthesis such as the tissue constructs describedin U.S. patent application Ser. No. 10/068,212 filed Feb. 6, 2002, canbe used.

As discussed above, when the vessel exhibits a defect such as thatdescribed above for venous valvular dysfunction, the construct orprosthesis can serve to modify the shape of the vessel in the region ofthe leaflet bases. This, in turn, can reduce the interior dimension ofthe vessel in the direction extending from one leaflet base to another.This in turn can improve the function of the valve, e.g. by improvingthe co-aptation of the opposing leaflets of the valve.

A sufficient amount of the remodelable biomaterial is used to treat thevessel. In certain embodiments, a sufficient amount of material is usedto encircle greater than about 20% of the circumference of the vessel;more preferably, a sufficient amount of the remodelable biomaterial isused to encircle greater than about 60% of the vessel; still morepreferable, greater than about 80% of the vessel. In certainembodiments, an effective amount of the biomaterial is implanted aboutthe vessel to reinforce the vessel wall, and/or to modify the diameteror shape of the vessel wall. Still further, in certain embodiments, thebiomaterial is implanted to modify the function of the valve within thevessel.

In some embodiments of the invention, the biomaterial is selected so asto retract or shrink after implantation in vivo. For instance, as aremodelable material retracts upon remodeling, it can press against orcompress the periphery of the vessel. The remodelable material can beprepared to shrink at varying rates and to varying extents as desired.The shrinkage can also be controlled by a judicious selection of thesource of the biomaterial and its preparation. For example thebiomaterial can include a two or more layers, which are layers can beobtained from different tissue or obtained from the same tissue type butprepared differently. Alternatively or in addition, the selectedbiomaterial may exhibit anisotropic shrinkage, e.g. upon remodeling withhost tissue. Consequently, the biomaterial shrinks to a greater extentin a first direction while exhibiting relatively less shrinkage in asecond direction. Two or more layers of such a biomaterial either aloneor in conjunction with other layers can be laminated such that theanisotropic shrinkage affects of the layers either complements eachother, e.g. as in multilaminate materials having the layers orientedwith their directions of maximal shrinkage generally aligned, orinterferes with or modifies each other, e.g. as in multilaminatematerials having the layers oriented with their directions of maximalshrinkage non-aligned or generally transverse to one another.

The biomaterial can also be crosslinked to vary and/or control theextent and/or rate of shrinkage. Increasing the amount (or number) ofcrosslinkages within the biomaterial or between two or more layers ofthe construct can be used to decrease the relative amount of shrinkage.However, crosslinkages within the biomaterial may also effect itsremodelability. Consequently, in applications where tissue remodeling isdesired, the biomaterial may substantially retain its native level ofcrosslinking, or the amount of added crosslinkages within thebiomaterial can be judiciously selected depending upon the desiredtreatment regime. In many cases, the biomaterial will exhibitremodelable properties such that the remodeling process occurs over thecourse of several days or several weeks. In preferred embodiments, theremodeling process occurs within a matter of about 5 days to about 12weeks.

For use in the present invention, introduced crosslinking of thebiomaterial may be achieved by photo-crosslinking techniques, bychemical crosslinkers, or by protein crosslinking induced by dehydrationor other means. Chemical crosslinkers that may be used include forexample aldehydes such as glutaraldehydes, diimides such ascarbodiimides, e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride, ribose or other sugars, acyl-azide,sulfo-N-hydroxysuccinamide, or polyepoxide compounds, including forexample polyglycidyl ethers such as ethyleneglycol diglycidyl ether,available under the trade name DENACOL EX810 from Nagese Chemical Co.,Osaka, Japan, and glycerol polyglycerol ether available under the tradename DENACOL EX 313 also from Nagese Chemical Co. Typically, when used,polyglycerol ethers or other polyepoxide compounds will have from 2 toabout 10 epoxide groups per molecule.

When a laminate of material is used in the present invention, the layersof the laminate can be additionally crosslinked to bond multiplesubmucosa layers to one another. Thus, additional crosslinking may beadded to individual submucosa layers prior to bonding to one another,during bonding to one another, and/or after bonding to one another.

In some embodiments of the invention, the prosthesis is prepared using atwo component bonding agent such as fibrin glue (e.g., having thrombinand fibrinogen as separate components). To prepare such prostheses,subsequent layers are added after coating the previously-applied layerwith a first component of the bonding agent (e.g., thrombin) and coatinga layer to be applied with a second component of the bonding agent(e.g., fibrinogen). Thereafter, the layer to be applied is positionedover the previously-applied layer so as to bring the two bondingcomponents into contact, thus causing the curing process to begin. Thisprocess can be repeated for any and all additional layers in a laminatedconstruct. Additionally this process can be used to bond the ends of aprosthesis together in vivo.

In another mode of treatment, an injectable or otherwise flowablebiocompatible material can be introduced in the patient to treat avascular vessel site associated with a valve. For example, such acomposition can be introduced into the vessel wall, e.g. into or inbetween one or more coatings of the vessel wall, and/or can beintroduced into an external region surrounding a vessel wall, e.g. incontact with the external connective tissue coating (tunica adventitia)of an vein or artery. The injectable or flowable composition can beintroduced at a single site or at a plurality of sites within the wallof a vascular vessel and/or about the external periphery of the vascularvessel. Relatively non-invasive modes of introduction will beadvantageous, e.g. involving the delivery of the flowable compositionthrough a cannulated device such as a needle or catheter.

In certain embodiments of the invention, an effective amount of aflowable composition is injected or otherwise introduced so as toprovide support for and/or increase the strength of vessel walls. Theintroduced composition can include one or more bioactive agents orviable cellular populations, or combinations of these. Non-limitingexamples of bioactive agents are listed above. Preferably the bioactiveagents are effective to promote tissue growth.

While other biocompatible materials that can provide bulk mass toreinforce or modify the shape of the vessel walls may be used, theinjectable or flowable composition for use in the invention desirablycomprises an ECM composition. For example, fluidized submucosa can beprepared as described in U.S. Pat. Nos. 5,275,826 and 6,444,229.Fluidized or otherwise flowable ECM compositions may also be prepared asdescribed in co-pending International Patent Application No.PCT/US04/27557 filed Aug. 25, 2004 and entitled Graft MaterialsContaining Bioactive Substances, and Methods for Their Manufacture. Inthis regard, the flowable compositions of the invention can be preparedfrom an isolated ECM material, for example one of those listed above.The ECM material is used to prepare a solubilized mixture includingcomponents of the material. This can be achieved by digestion of the ECMmaterial in an acidic or basic medium and/or by contact with anappropriate enzyme or combination of enzymes.

The ECM material can be reduced to particulate form to aid in thedigestion step. This can be achieved by tearing, cutting, grinding orshearing the isolated ECM material. Illustratively, shearing may beconducted in a fluid medium, and grinding may be conducted with thematerial in a frozen state. For example, the material can be contactedwith liquid nitrogen to freeze it for purposes of facilitating grindinginto powder form. Such techniques can involve freezing and pulverizingsubmucosa under liquid nitrogen in an industrial blender.

Any suitable enzyme may be used for an enzymatic digestion step. Suchenzymes include for example serine proteases, aspartyl proteases, andmatrix metalloproteases. The concentration of the enzyme can be adjustedbased on the specific enzyme used, the amount of submucosa to bedigested, the duration of the digestion, the temperature of thereaction, and the desired properties of the final product. In oneembodiment about 0.1% to about 0.2% of enzyme (pepsin, for example) isused and the digestion is conducted under cooled conditions for a periodof time sufficient to substantially digest the ECM material. Thedigestion can be conducted at any suitable temperature, withtemperatures ranging from 4-37° C. being preferred. Likewise, anysuitable duration of digestion can be used, such durations typicallyfalling in the range of about 2-180 hours. The ratio of theconcentration of ECM material (hydrated) to total enzyme usually rangesfrom about 25 to about 125 and more typically the ratio is about 50, andthe digestion is conducted at 4° C. for 24-72 hours. When an enzyme isused to aid in the digestion, the digestion will be performed at a pH atwhich the enzyme is active and more advantageously at a pH at which theenzyme is optimally active. Illustratively, pepsin exhibits optimalactivity at pH's in the range of about 2-4.

The enzymes or other disruptive agents used to solubilize the ECMmaterial can be removed or inactivated before or during the gellingprocess so as not to compromise gel formation or subsequent gelstability. Also, any disruptive agent, particularly enzymes, thatremains present and active during storage of the tissue will potentiallychange the composition and potentially the gelling characteristics ofthe solution. Enzymes, such as pepsin, can be inactivated with proteaseinhibitors, a shift to neutral pH, a drop in temperature below 0° C.,heat inactivation or through the removal of the enzyme by fractionation.A combination of these methods can be utilized to stop digestion of theECM material at a predetermined endpoint, for example the ECM materialcan be immediately frozen and later fractionated to limit digestion.

The ECM material is enzymatically digested for a sufficient time toproduce a hydrolysate of ECM components. The ECM can be treated with oneenzyme or with a mixture of enzymes to hydrolyze the structuralcomponents of the material and prepare a hydrolysate having multiplehydrolyzed components of reduced molecular weight. The length ofdigestion time is varied depending on the application, and the digestioncan be extended to completely solubilize the ECM material. In some modesof operation, the ECM material will be treated sufficiently to partiallysolubilize the material to produce a digest composition comprisinghydrolyzed ECM components and nonhydrolyzed ECM components. The digestcomposition can then optionally be further processed to remove at leastsome of the nonhydrolyzed components. For example, the nonhydrolyzedcomponents can be separated from the hydrolyzed portions bycentrifugation, filtration, or other separation techniques known in theart.

Preferred gel compositions of the present invention are prepared fromenzymatically digested vertebrate ECM material that has beenfractionated under acidic conditions, for example including pH rangingfrom about 2 to less than 7, especially to remove low molecular weightcomponents. Typically, the ECM hydrolysate is fractionated by dialysisagainst a solution or other aqueous medium having an acidic pH, e.g. apH ranging from about 2 to about 5, more desirably greater than 3 andless than 7. In addition to fractionating the hydrolysate under acidicconditions, the ECM hydrolysate is typically fractionated underconditions of low ionic strength with minimal concentrations of saltssuch as those usually found in standard buffers such as PBS (i.e. NaCl,KCl, Na2HPO4, or KH2PO4) that can pass through the dialysis membrane andinto the hydrolysate. Such fractionation conditions work to reduce theionic strength of the ECM hydrolysate and thereby provide enhanced gelforming characteristics.

The hydrolysate solution produced by enzymatic digestion of the ECMmaterial has a characteristic ratio of protein to carbohydrate. Theratio of protein to carbohydrate in the hydrolysate is determined by theenzyme utilized in the digestion step and by the duration of thedigestion. The ratio may be similar to or may be substantially differentfrom the protein to carbohydrate ratio of the undigested ECM tissue. Forexample, digestion of vertebrate ECM material with a protease such aspepsin, followed by dialysis, will form a fractionated ECM hydrolysatehaving a lower protein to carbohydrate ratio relative to the originalECM material.

Flowable ECM compositions capable of forming shape retaining gels can beused in the present invention. Such ECM compositions can be preparedfrom ECM material that has been enzymatically digested and fractionatedunder acidic conditions to form an ECM hydrolysate that has a protein tocarbohydrate ratio different than that of the original ECM material.Such fractionation can be achieved entirely or at least in part bydialysis. The molecular weight cut off of the ECM components to beincluded in the gellable material is selected based on the desiredproperties of the gel. Typically the molecular weight cutoff of thedialysis membrane (the molecular weight above which the membrane willprevent passage of molecules) is within in the range of about 2000 toabout 10000 Dalton, and more preferably from about 3500 to about 5000Dalton.

In certain forms of the gellable ECM composition, apart from thepotential removal of undigested ECM components after the digestion stepand any controlled fractionation to remove low molecular weightcomponents as discussed above, the ECM hydrolysate is processed so as toavoid any substantial further physical separation of the ECM components.For example, when a more concentrated ECM hydrolysate material isdesired, this can be accomplished by removing water from the system(e.g. by evaporation or lyophilization) as opposed to using conventional“salting out”/centrifugation techniques that would demonstratesignificant selectivity in precipitating and isolating collagen, leavingbehind amounts of other desired ECM components. Thus, in certainembodiments of the invention, solubilized ECM components of the ECMhydrolysate remain substantially unfractionated, or remain substantiallyunfractionated above a predetermined molecular weight cutoff such asthat used in the dialysis membrane, e.g. above a given value in therange of about 2000 to 10000 Dalton, more preferably about 3500 to about5000 Dalton.

Vertebrate ECM material can be stored frozen (e.g. at about −20 toabout−80° C.) in either its solid, comminuted or enzymatically digestedforms, or the material can be stored after being hydrolyzed andfractionated. The ECM material can be stored in solvents that maintainthe collagen in its native form and solubility. For example, onesuitable storage solvent is 0.01 M acetic acid, however other acids canbe substituted, such as 0.01 N HCl. In one form, the fractionated ECMhydrolysate can be dried (by lyophilization, for example) and stored ina dehydrated/lyophilized state. The dried form can be rehydrated toprepare a flowable ECM composition capable of forming a gel.

In accordance with one embodiment, the fractionated ECM hydrolysate orother flowable ECM composition will exhibit the capacity to gel uponadjusting the pH of a relatively more acidic aqueous medium containingit to about 5 to about 9, more preferably about 6.6 to about 8.0, andtypically about 7.2 to about 7.8, thus inducing fibrillogenesis andmatrix gel assembly. In one embodiment, the pH of the fractionatedhydrolysate is adjusted by the addition of a buffer that does not leavea toxic residue, and has a physiological ion concentration and thecapacity to hold physiological pH. Examples of suitable buffers includePBS, HEPES, and DMEM. Illustratively, the pH of the fractionated ECMhydrolysate can be raised by the addition of a buffered NaOH solution to6.6 to 8.0, more preferably 7.2 to 7.8, to facilitate the formation ofan ECM-containing gel. Any suitable concentration of NaOH solution canbe used for these purposes, for example including about 0.05 M to about0.5 M NaOH. In accordance with one embodiment, the ECM hydrolysate ismixed with a buffer and sufficient 0.25 N NaOH is added to the mixtureto achieve the desired pH.

The ionic strength of the ECM hydrolysate is believed to be important inmaintaining the fibers of collagen in a state that allows forfibrillogenesis and matrix gel assembly upon neutralization of thehydrolysate. Accordingly, if needed, the salt concentration of the ECMhydrolysate material can be reduced prior to neutralization of thehydrolysate. The neutralized hydrolysate can be caused to gel at anysuitable temperature, e.g. ranging from about 4° C. to about 40° C. Thetemperature will typically affect the gelling times, which may rangefrom 5 to 120 minutes at the higher gellation temperatures and 1 to 8hours at the lower gellation temperatures. Typically, the hydrolysatewill be effective to self-gel at elevated temperatures, for example atabout 37° C. In this regard, preferred neutralized ECM hydrolysates willbe effective to gel in less than about ninety minutes at 37° C., forexample approximately thirty to ninety minutes at 37° C.

Additional components can be added to the ECM hydrolysate compositionbefore, during or after forming the gel. For example, proteinscarbohydrates, growth factors, therapeutics, bioactive agents, nucleicacids, cells or pharmaceuticals can be added. In certain embodiments,such materials are added prior to formation of the gel. This may beaccomplished for example by forming a dry mixture of a powdered ECMhydrolysate with the additional component(s), and then reconstitutingand gelling the mixture, or by incorporating the additional component(s)into an aqueous, ungelled composition of the ECM hydrolysate before,during (e.g. with) or after addition of the neutralization agent. Theadditional component(s) can also be added to the formed ECM gel, e.g. byinfusing or mixing the component(s) into the gel and/or coating themonto the gel.

In one embodiment of the invention, a particulate ECM material will beadded to the ECM hydrolysate composition, which will then beincorporated in the formed gel. Such particulate ECM materials can beprepared by cutting, tearing, grinding or otherwise comminuting an ECMstarting material. For example, a particulate ECM material having anaverage particle size of about 50 microns to about 500 microns may beincluded in the gellable ECM hydrolysate, more preferably about 100microns to about 400 microns. The ECM particulate can be added in anysuitable amount relative to the hydrolysate, with preferred ECMparticulate to ECM hydrolysate weight ratios (based on dry solids) beingabout 0.1:1 to about 200:1, more preferably in the range of 1:1 to about100:1. The inclusion of such ECM particulates in the ultimate gel canserve to provide additional material that can function to providebioactivity to the gel (e.g. itself including FGF-2 and/or other growthfactors or bioactive substances as discussed herein) and/or serve asscaffolding material for tissue ingrowth.

In certain embodiments, an ECM hydrolysate material to be used in theinvention will exhibit an injectable character and also incorporate anECM particulate material. In these embodiments, the ECM particulatematerial can be included at a size and in an amount that effectivelyretains an injectable character to the hydrolysate composition, forexample by injection through a needle having a size in the range of 18to 31 gauge (internal diameters of 0.047 inches to about 0.004 inches).In this fashion, non-invasive procedures for tissue augmentation will beprovided, which in preferred cases will involve the injection of anungelled ECM hydrolysate containing suspended ECM particles at arelatively lower (e.g. room) temperature, which will be promoted to forma gelled composition when injected into a patient and thereby brought tophysiologic temperature (about 37° C.).

In certain embodiments, flowable ECM compositions to be used in theinvention may be disinfected by contacting an aqueous medium includingECM hydrolysate components with an oxidizing disinfectant. This mode ofdisinfection provides an improved ability to recover a disinfected ECMhydrolysate that exhibits the capacity to form beneficial gels. Incertain preparative methods, an aqueous medium containing ECMhydrolysate components can disinfected by providing a peroxydisinfectant in the aqueous medium. This can be advantageously achievedusing dialysis to deliver the peroxy disinfectant into and/or to removethe peroxy disinfectant from the aqueous medium containing thehydrolysate. In certain dinsinfection techniques, an aqueous mediumcontaining the ECM hydrolysate is dialyzed against an aqueous mediumcontaining the peroxy disinfectant to deliver the disinfectant intocontact with the ECM hydrolysate, and then is dialyzed against anappropriate aqueous medium (e.g. an acidic aqueous medium) to at leastsubstantially remove the peroxy disinfectant from the ECM hydrolysate.During this dialysis step, the peroxy compound passes through thedialysis membrane and into the ECM hydrolysate, and contacts ECMcomponents for a sufficient period of time to disinfect the ECMcomponents of the hydrolysate. In this regard, typical contact timeswill range from about 0.5 hours to about 8 hours and more typicallyabout 1 hour to about 4 hours. The period of contact will be sufficientto substantially disinfect the digest, including the removal ofendotoxins and inactivation of virus material present. The removal ofthe peroxy disinfectant by dialysis may likewise be conducted over anysuitable period of time, for example having a duration of about 4 toabout 180 hours, more typically about 24 to about 96 hours. In general,the disinfection step will desirably result in a disinfected ECMhydrolysate composition having sufficiently low levels of endotoxins,viral burdens, and other contaminant materials to render it suitable formedical use. Endotoxin levels below about 2 endotoxin units (EUs) pergram (dry weight) are preferred, more preferably below about 1 EU pergram, as are virus levels below 100 plaque forming units per gram (dryweight), more preferably below 1 plaque forming unit per gram.

The aqueous ECM hydrolysate composition can be a substantiallyhomogeneous solution during the dialysis step for delivering theoxidizing disinfectant to the hydrolysate composition and/or during thedialysis step for removing the oxidizing disinfectant from thehydrolysate composition. Alternatively, the aqueous hydrolysatecomposition can include suspended ECM hydrolysate particles, optionallyin combination with some dissolved ECM hydrolysate components, duringeither or both of the oxidizing disinfectant delivery and removal steps.Dialysis processes in which at least some of the ECM hydrolysatecomponents are dissolved during the disinfectant delivery and/or removalsteps are preferred and those in which substantially all of the ECMhydrolysate components are dissolved are more preferred.

The disinfection step can be conducted at any suitable temperature, andwill typically be conducted between 0° C. and 37° C., more typicallybetween about 4° C. and about 15° C. During this step, the concentrationof the ECM hydrolysate solids in the aqueous medium can be in the rangeof about 2 mg/ml to about 200 mg/ml, and may vary somewhat through thecourse of the dialysis due to the migration of water through themembrane. In certain embodiments, a relatively unconcentrated digest isused, having a starting ECM solids level of about 5 mg/ml to about 15mg/ml. In other embodiments, a relatively concentrated ECM hydrolysateis used at the start of the disinfection step, for example having aconcentration of at least about 20 mg/ml and up to about 200 mg/ml, morepreferably at least about 100 mg/ml and up to about 200 mg/ml. It hasbeen found that the use of concentrated ECM hydrolysates during thisdisinfection processing results in an ultimate gel composition havinghigher gel strength than that obtained using similar processing with alower concentration ECM hydrolysate. Accordingly, processes whichinvolve the removal of amounts of water from the ECM hydrolysateresulting from the digestion prior to the disinfection processing stepare preferred. For example, such processes may include removing only aportion of the water (e.g. about 10% to about 98% by weight of the waterpresent) prior to the dialysis/disinfection step, or may includerendering the digest to a solid by drying the material by lyophilizationor otherwise, reconstituting the dried material in an aqueous medium,and then treating that aqueous medium with the dialysis/disinfectionstep.

In one mode of operation, the disinfection of the aqueous mediumcontaining the ECM hydrolysate can include adding the peroxy compound orother oxidizing disinfectant directly to the ECM hydrolysate, forexample being included in an aqueous medium used to reconstitute a driedECM hydrolysate or being added directly to an aqueous ECM hydrolysatecomposition. The disinfectant can then be allowed to contact the ECMhydrolysate for a sufficient period of time under suitable conditions(e.g. as described above) to disinfect the hydrolysate, and then removedfrom contact with the hydrolysate. In one embodiment, the oxidizingdisinfectant can then be removed using a dialysis procedure as discussedabove. In other embodiments, the disinfectant can be partially orcompletely removed using other techniques such as chromatographic or ionexchange techniques, or can be partially or completely decomposed tophysiologically acceptable components. For example, when using anoxidizing disinfectant containing hydrogen peroxide (e.g. hydrogenperoxide alone or a peracid such as peracetic acid), hydrogen peroxidecan be allowed or caused to decompose to water and oxygen, for examplein some embodiments including the use of agents that promote thedecomposition such as thermal energy or ionizing radiation, e.g.ultraviolet radiation.

In another mode of operation, the oxidizing disinfectant can bedelivered into the aqueous medium containing the ECM hydrolysate bydialysis and processed sufficiently to disinfect the hydrolysate (e.g.as described above), and then removed using other techniques such aschromatographic or ion exchange techniques in whole or in part, orallowed or caused to decompose in whole or in part as discussedimmediately above.

Peroxygen compounds that may be used in the disinfection step include,for example, hydrogen peroxide, organic peroxy compounds, and preferablyperacids. Such disinfecting agents are used in a liquid medium,preferably a solution, having a pH of about 1.5 to about 10.0, moredesirably about 2.0 to about 6.0. As to peracid compounds that can beused, these include peracetic acid, perpropioic acid, or perbenzoicacid. Peracetic acid is the most preferred disinfecting agent forpurposes of the present invention.

When used, peracetic acid is desirably diluted into about a 2% to about50% by volume of alcohol solution, perferably ethanol. The concentrationof the peracetic acid may range, for instance, from about 0.05% byvolume to about 1.0% by volume. Most preferably, the concentration ofthe peracetic acid is from about 0.1% to about 0.3% by volume. Whenhydrogen peroxide is used, the concentration can range from about 0.05%to about 30% by volume. More desirably the hydrogen peroxideconcentration is from about 1% to about 10% by volume, and mostpreferably from about 2% to about 5% by volume. The solution may or maynot be buffered to a pH from about 5 to about 9, with more preferredpH's being from about 6 to about 7.5. These concentrations of hydrogenperoxide can be diluted in water or in an aqueous solution of about 2%to about 50% by volume of alcohol, most preferably ethanol. Additionalinformation concerning preferred peroxy disinfecting agents can be foundin discussions in U.S. Pat. No. 6,206,931, which is herein incorporatedby reference.

Flowable ECM materials of the present invention can be prepared to havedesirable properties for handling and use. For example, fluidized ECMhydrolysates can be prepared in an aqueous medium, which can thereafterbe effective to form of a gel. Such prepared aqueous mediums can haveany suitable level of ECM hydrolysate therein. Typically, the ECMhydrolysate will be present in the aqueous medium at a concentration ofabout 2 mg/ml to about 200 mg/ml, more typically about 20 mg/ml to about200 mg/ml, and in some embodiments about 30 mg/ml to about 120 mg/ml. Inpreferred forms, the aqueous ECM hydrolysate composition will have aninjectable character, for example by injection through a needle having asize in the range of 18 to 31 gauge (internal diameters of about 0.047inches to about 0.004 inches).

Furthermore, flowable ECM compositions can be prepared so that inaddition to neutralization, heating to physiologic temperatures (such as37° C.) will substantially reduce the gelling time of the material. Itis contemplated that commercial products or systems of the invention andfor practice of treatments of the invention may include (i) packaged,sterile powders which can be reconstituted in an acidic medium to form aflowable ECM composition, e.g. one which gels when neutralized andpotentially heated; (ii) packaged, sterile aqueous compositionsincluding solubilized ECM hydrolysate components under non-gelling (e.g.acidic) conditions; or (iii) packaged, sterile ECM gel compositions. Incertain embodiments, a medical kit for performing a treatment of avascular vessel in accordance with the invention is provided thatincludes a packaged, sterile aqueous composition including solubilizedECM hydrolysate components under non-gelling (e.g. acidic) conditions,and a separately packaged, sterile aqueous neutralizing composition(e.g. containing a buffer and/or base) that is adapted to neutralize theECM hydrolysate medium for the formation of a gel. In anotherembodiment, a medical kit for treatments of the invention includes apackaged, sterile, dried (e.g. lyophilized) ECM hydrolysate powder, aseparately packaged, sterile aqueous acidic reconstituting medium, and aseparately packaged sterile, aqueous neutralizing medium. In use, theECM hydrolysate powder can be reconstituted with the reconstitutingmedium to form a non-gelled mixture, which can then be neutralized withthe neutralizing medium. The thus-neutralized flowable ECM material canthen be delivered to a treatment site in accordance with the inventionbefore or after it gels.

Medical kits as described above may also include a device, such as asyringe, for delivering the neutralized ECM hydrolysate medium to apatient. In this regard, the sterile, aqueous ECM hydrolysate medium orthe sterile ECM hydrolysate powder of such kits can be provided packagedin a syringe or other delivery instrument. In addition, the sterilereconstituting and/or neutralizing medium can be packaged in a syringe,and means provided for delivering the contents of the syringe into toanother syringe containing the aqueous ECM hydrolysate medium or the ECMhydrolysate powder for mixing purposes. In still other forms of theinvention, a self-gelling aqueous ECM hydrolysate composition can bepackaged in a container (e.g. a syringe) and stable against gelformation during storage. For example, gel formation of such productscan be dependent upon physical conditions such as temperature or contactwith local milieu present at an implantation site in a patient.Illustratively, an aqueous ECM hydrolysate composition that does not gelor gels only very slowly at temperatures below physiologic temperature(about 37° C.) can be packaged in a syringe or other container andpotentially cooled (including for example frozen) prior to use forinjection or other implantation into a patient for a treatment inaccordance with the invention.

The manipulations used to prepare ECM hydrolysate compositions andgellable or gelled forms thereof can also have a significant impact upongrowth factors or other ECM components that may contribute tobioactivity. Techniques for modulating and sampling for levels of FGF-2or other growth factors or bioactive substances can be used inconjunction with the manufacture of the described ECM hydrolysatecompositions. Illustratively, the dialysis/disinfection processesdescribed above employing peroxy compounds typically cause a reductionin the level of FGF-2 in the ECM hydrolysate material. In work to dateas described in Examples 1-4, such processing using peracetic acid asdisinfectant has caused a reduction in the level of FGF-2 in the rangeof about 30% to about 50%. Accordingly, to retain higher levels ofFGF-2, one can process for a minimal about of time necessary to achievethe desired disinfection of the material; on the other hand, to reducethe FGF-2 to lower levels, the disinfection processing can be continuedfor a longer period of time.

In certain methods of manufacturing a flowable ECM composition, thedisinfection process and subsequent steps will be sufficiently conductedto result in a medically sterile aqueous ECM hydrolysate composition,which can be packaged using sterile filling operations. In othermanufacturing methods, any terminal sterilization applied to the ECMhydrolysate material (e.g. in dried powder, non-gelled aqueous medium,or gelled form) can also be selected and controlled to optimize thelevel of FGF-2 or other bioactive substances in the product. Terminalsterilization methods may include, for example, high or low temperatureethylene oxide, radiation such as E-beam, gas plasma (e.g. Sterrad), orhydrogen peroxide vapor processing.

Preferred, packaged, sterilized ECM hydrolysate products for use inaccordance with the invention will have an FGF-2 level (this FGF-2 beingprovided by the ECM hydrolysate) of about 100 ng/g to about 5000 ng/gbased upon the dry weight of the ECM hydrolysate. More preferably, thisvalue will be about 300 ng/g to about 4000 ng/g. As will be understood,such FGF-2 levels can be determined using standard ELISA tests (e.g.using the Quantikine Human Basic Fibroblast Growth Factor ELISA kitcommercially available from R&D Systems).

With reference now to FIGS. 8 and 9, in one illustrative embodiment ofthe invention, all or a portion of a vein 90 in the region of a valve 92is surrounded by a flowable composition 94. Flowable composition 94 isdesirably one that promotes the growth of tissue in the patient, and isdesirably a flowable tissue graft material such as a flowable ECMcomposition. In certain embodiments, the flowable composition isinjected about the periphery of the vessel, and allowed or caused to gelor otherwise harden to provide a cuff 96 of material suitable tosupplement or support the vessel wall. Still further, in someembodiments of the invention, the cuff 96 can exhibit retraction orcontraction upon ingrowth of patient tissue, so as to modify the shapeof the vessel in the cuffed region, e.g. to improve the function of theinterior valve. In certain embodiments, the flowable material isintroduced by injection using a cannulated percutaneous device 98 suchas one having needle, which is used to puncture and exit the vein 90 atone or more sites and upon doing so is used to deliver the flowablecomposition through the cannula to surround all or a portion of theexterior of the vein 90. Alternatively or in addition, an externallydelivered needle 100 can be used to deliver the flowable composition tothe exterior periphery of the vein 90. The flowable mass, whichoptionally gels or otherwise hardens on its own or can be induced to gelor harden, can serve to reinforce the walls of the vein 90 and/or toapply pressure to and re-shape the walls of the vein to modify andimprove the function of the valve 92. In certain embodiments, theflowable mass can be a remodelable ECM biomaterial or another materialthat promotes tissue ingrowth. In still further embodiments, such tissueingrowth can lead to retraction or shrinkage of the introduced mass asit is replaced by host tissue, thus serving to promote close contact ofthe ingrown tissue with the vein wall and/or to re-shape the vein walland potentially modify the function of the interior valve 92.

FIG. 10 provides an illustration of a vein 110 having an interior valve112 and associated vein walls 114 into which a therapeutic composition116 has been introduced. Composition 116, may, for example, be effectiveto promote tissue growth, and may be a flowable biomaterial as discussedherein. Composition 116 can be delivered through a percutaneous needledevice 118, which can be manipulated to one or multiple positions (seephantom) to deliver composition 116 into the vein walls 114 in theregion of valve 112. Composition 116 and potential tissue developmentstimulated by composition 116 can serve to strengthen vein walls 114,for example to protect against initial or continued distension. Inaddition or alternatively, localized retraction of vein walls 114stimulated by the introduction of composition 116 can serve to re-shapethe vein walls, for example to impact and improve upon the function ofvalve 112.

The prosthesis and biomaterial either as a construct or as a fluidizedor flowable substance can optionally be rendered radiopaque in whole orin part by a variety of methods as discussed in WO 00/32112, which isincorporated herein. In one embodiment for the invention, when thebiomaterial is formed into sheets whether in lyophilized ornon-lyophilized form a radiopaque substance including but not limitedto, tantalum such as tantalum powder, can be spread along the surface ofthe layers. When the prosthesis or biomaterial is provided as a flowablecomposition, the radiopaque substance can be combined either with thepowdered form or as the hydrated/rehydrated form prior to or duringtreatment. Other radiopaque agents for use in this invention includebismuth, iodine, and barium as well as other conventional markers. Itwill be understood that the radiopaque substance may be incorporatedhomogeneously or inhomogeneously within or on the biomaterial to beimplanted.

Embodiments of the invention have been discussed above generally inconnection with treating vascular vessels at regions associated withnative valve structures. In other embodiments, the inventive methods,constructs and materials as described above can be used in thestrengthening and/or reinforcement of vascular vessel walls in regionsproximate to an introduced framed or frameless artificial vascularvalve. As well, such strengthening or reinforcement techniques can beused in conjunction with other cut-down surgical or percutaneoustreatments to improve an existing native valve, for example bymanipulating or modifying the valve leaflets themselves. Certainembodiments of the invention, accordingly, involve patient treatmentregimens which include treating a native valve or implanting anartificial valve in a vascular vessel in combination with strengtheningor reinforcing walls of the vessel in affected or potentially affectedregions associated with the treated or implanted valve. In morepreferred embodiments, the vascular vessel is a vein, and the valve is avenous valve. In especially preferred aspects, veins within the legs orfeet will be treated in accordance with the invention.

The invention also encompasses medical products for use in treatments ofthe invention, which products include a prosthesis device or flowablebiocompatible material sealed within sterile medical packaging. Thefinal, packaged product is provided in a sterile condition. This may beachieved, for example, by gamma, e-beam or other irradiation techniques,ethylene oxide gas, or any other suitable sterilization technique, andthe materials and other properties of the medical packaging will beselected accordingly.

In order to promote a further understanding of the present invention andits features and advantages, the following specific examples areprovided. It will be understood that these examples are illustrative andare not limiting of the invention.

EXAMPLE 1

Raw (isolated/washed but non-disinfected) porcine small intestinesubmucosa was frozen, cut into pieces, and cryoground to powder withliquid nitrogen. 50 g of the submucosa powder was mixed with one literof a digestion solution containing 1 g of pepsin and 0.5 M acetic acid.The digestion process was allowed to continue for 48-72 hours underconstant stirring at 4° C. At the end of the process, the digest wascentrifuged to remove undigested material. The acetic acid was thenremoved by dialysis against 0.01 M HCl for approximately 96 hours at 4°C. The resulting digest was transferred (without concentration) into asemipermeable membrane with a molecular weight cut off of 3500, anddialyzed for two hours against a 0.2 percent by volume peracetic acid ina 5 percent by volume aqueous ethanol solution at 4° C. This step servedboth to disinfect the submucosa digest and to fractionate the digest toremove components with molecular weights below 3500. The PAA-treateddigest was then dialysed against 0.01 M HCl for 48 hours at 4° C. toremove the peracetic acid. The sterilized digest was concentrated bylyophilization, forming a material that was reconstituted at about 30mg/ml solids in 0.01 M HCl. This material was neutralized with phosphatebuffered NaOH to a pH of about 7.5-7.6 which provided a flowablematerial which formed a gel when heated to physiologic temperature.

EXAMPLE 2

A second acetic acid processed submucosa gel was made using a processsimilar to that described in Example 1 above, except concentrating thedigest prior to the PAA treatment. Specifically, immediately followingthe removal of acetic acid by dialysis, the digest was lyophilized todryness. A concentrated paste of the digest was made by dissolving apre-weighed amount of the lyophilized product in a known amount of 0.01M HCl to prepare a mixture having an ECM solids concentration of about50 mg/ml. The concentrated paste was then dialysed against the PAAsolution for 2 hours and then against 0.01 M HCl for removal of PAA inthe same manner described in Example 1. The digest was adjusted to about30 mg/ml solids and neutralized with phosphate buffered NaOH to a pH ofabout 7.5-7.6, which upon heating to physiologic temperature formed agel.

EXAMPLE 3

An HCl processed submucosa gel was made using a procedure similar tothat described in Example 1, except using 0.01 M of HCl in thepepsin/digestion solution rather than the 0.5 M of acetic acid, andomitting the step involving removal of acetic acid since none waspresent. The digest was used to form a gel as described in Example 1.

EXAMPLE 4

Another HCl processed submucosa gel was made using a procedure similarto that described in Example 2, except using 0.01 M of HCl in thepepsin/digestion solution rather than the 0.5 M of acetic acid, andomitting the step involving removal of acetic acid since none waspresent. The digest was used to form a gel as described in Example 2.

The present invention contemplates other modifications as would occur tothose skilled in the art. It is also contemplated that methodologiesembodied in the present invention can be altered or added to otherprocesses as would occur to those skilled in the art without departingfrom the spirit of the present invention. All publications, patents, andpatent applications cited in this specification are herein incorporatedby reference as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference and set forth in its entirety herein.

Further, any theory of operation, proof, or finding stated herein ismeant to further enhance understanding of the present invention and isnot intended to make the scope of the present invention dependent uponsuch theory, proof, or finding.

1. A method for treating a vein in a patient, said method comprising:accessing a treatment site proximate to a valve in the vein, introducinga remodelable biomaterial into, or in contact with an exterior surfaceof, a wall of the vein at the treatment site, and wherein saidremodelable biomaterial is effective to shrink as a result of remodelingso as to reshape the vein.
 2. The method of claim 1 wherein the valve isan insufficient valve.
 3. The method of claim 1 wherein said introducingincludes introducing the remodelable biomaterial external of a wall ofthe vein.
 4. The method of claim 1 wherein said introducing includesintroducing the remodelable biomaterial into a wall of the vein.
 5. Themethod of claim 1 wherein the remodelable biomaterial comprises anextracellular matrix material.
 6. The method of claim 1 wherein theremodelable biomaterial comprises submucosa tissue.
 7. The method ofclaim 1 wherein the remodelable biomaterial comprises collagen.
 8. Themethod of claim 6, wherein said submucosa is porcine, bovine, or ovinesubmucosa.
 9. The method of claim 1 wherein the remodelable biomaterialcomprises a bioactive substance.
 10. The method of claim 1 wherein theremodelable biomaterial comprises a population of viable cells.
 11. Themethod of claim 10 wherein the cells comprise autologous cells.
 12. Themethod of claim 1 wherein said introducing comprises wrapping a sheet ofthe remodelable biomaterial in contact with the external connectivetissue of the vein proximate to leaflet bases of the valve.
 13. Themethod of claim 12 comprising bonding the sheet of remodelablebiomaterial to itself to form a band about the external connectivetissue of the vein.
 14. The method of claim 1 wherein said introducingcomprises delivering an effective amount of the remodelable biomaterialexternally of the vein wall to reinforce the vein wall.
 15. The methodof claim 1 wherein the remodelable biomaterial is a laminate comprisingtwo or more sheets of a collagenous material.
 16. A method of treating avein of a patient, said method comprising: accessing a treatment sitealong the vein, said treatment site proximate to a valve within thevein; and introducing a flowable mass of biocompatible material incontact with an exterior surface of a wall of the vein at said treatmentsite.
 17. The method of claim 16 wherein the biocompatible material is acollagen-containing material.
 18. The method of claim 16, wherein saidflowable mass of biocompatible material is effective to re-shape thevein.
 19. The method of claim 16, wherein said flowable mass ofbiocompatible material is effective to reinforce the vein.
 20. Themethod of claim 16 wherein the biocompatible material comprises aremodelable biomaterial effective to retract upon remodeling so as tore-shape the vein.
 21. The method of claim 20 wherein the remodelablebiomaterial comprises an extracellular matrix material.
 22. The methodof claim 21 wherein the remodelable biomaterial comprises submucosa. 23.The method of claim 21, wherein the remodelable biomaterial comprises afluidized extracellular matrix material.
 24. The method of claim 21,wherein the remodelable biomaterial comprises a particulateextracellular matrix material.
 25. The method of claim 16 wherein thebiocompatible material comprises a population of viable cells.
 26. Themethod of claim 25, wherein said cells comprise autologous cells.
 27. Amethod for treating a vascular vessel in a patient, comprisingintroducing a remodelable biomaterial into, or in contact with anexterior surface of, a wall of the vessel in a region proximate to avascular valve in the vessel, wherein said remodelable biomaterial iseffective to shrink as a result of remodeling so as to re-shape thevessel.
 28. The method of claim 27 wherein said remodelable biomaterialhas a flowable state, and said introducing comprises injecting theremodelable biomaterial while in said flowable state.
 29. The method ofclaim 28 wherein said injecting includes injecting the remodelablebiomaterial into a site downstream of leaflet bases of the valve. 30.The method of claim 28 wherein said injecting includes injecting theremodelable biomaterial into a site upstream of leaflet bases of thevalve.
 31. The method of claim 28, wherein said injecting includesinjecting the remodelable biomaterial so as to contact an exteriorsurface of the vascular vessel in a region corresponding to leafletbases of the valve.
 32. The method of claim 28 wherein said injectingincludes injecting the remodelable biomaterial into external connectivetissue of the vascular vessel.
 33. The method of claim 28 wherein saidinjecting includes injecting the remodelable biomaterial into a middlemuscular layer of the vascular vessel.
 34. The method of claim 28wherein said injecting comprises injecting the remodelable biomaterialso as to substantially surround the vascular vessel.
 35. The method ofclaim 27 wherein the remodelable biomaterial comprises at least oneextracellular matrix material selected from the group consisting ofpericardium, submucosa, liver basement membrane, dura mater, peritoneum,serosa, and renal capsule.
 36. The method of claim 27, also comprisingintroducing a population of viable cells with the remodelablebiomaterial.
 37. The method of claim 36 wherein the cells compriseautologous cells.
 38. The method of claim 28 comprising injecting theremodelable biomaterial into two or more sites spaced circumferentiallyabout the vascular vessel.
 39. The method of claim 28 wherein theremodelable biomaterial also has a hardened state, and wherein saidmethod also comprises allowing or causing the remodelable biomaterial tochange from said flowable state to said hardened state after saidinjecting.
 40. The method of claim 39 wherein said remodelablebiomaterial is effective to be thermally induced to change from saidflowable state to said hardened state.
 41. The method of claim 40,wherein said remodelable biomaterial exhibits said flowable state at atemperature below the body temperature of the patient, and said hardenedstate at the body temperature of the patient.
 42. The method of claim 28wherein the remodelable biomaterial comprises a radiopaque substance.43. A method for treating a patient having an insufficient vascularvalve, said method comprising: establishing percutaneous access to avascular vessel of a patient; treating a native vascular valve and/orintroducing an artificial vascular valve in a vascular vessel of thepatient through the percutaneous access; and reinforcing the vascularvessel in a region proximate to said native vascular valve or artificialvascular valve by implanting a biocompatible material into a wall of thevessel or in contact with an exterior surface of a wall of the vessel.44. The method of claim 43 wherein the vascular vessel is a vein. 45.The method of claim 43 wherein said reinforcing comprises introducing aflowable biocompatible material in contact with exterior surface of awall of said vascular vessel.
 46. The method of claim 45 wherein theflowable biocompatible material promotes tissue growth in the patient.47. The method of claim 46 wherein the flowable biocompatible materialcomprises a remodelable material.
 48. The method of claim 47 wherein theflowable biocompatible material comprises an extracellular matrixmaterial.
 49. The method of claim 45 wherein the flowable biocompatiblematerial comprises a population of viable cells.
 50. The method of claim49 wherein the cells comprise autologous cells.